Derivatives of nucleosides, processes for the production of these derivatives of nucleosides and specific polyclonal and monoclonal antibodies of these derivatives

ABSTRACT

The invention relates to derivatives of nucleosides, processes for the production of these derivatives of nucleosides, as well as specific polyclonal and monoclonal antibodies of the aforementioned derivatives. 
     These derivatives comply with the following chemical formula: ##STR1## in which n is equal to 1, R 1  stands for H or a linear mono-, di- or tri-phosphoric acid, R 2  stands for a hydroxyl group, alkyl group, aryl group, a protein containing a free amino site, an aminoalkyl polystyrene or a silica grafted with an alkyl amine chain and R 3  represents a substituted base chosen from among uracil, thymine, cytosine, guanine or adenine.

The invention relates to derivatives of nucleosides, processes for theproduction of these derivatives of nucleosides, as well as specificpolyclonal and monoclonal antibodies of the aforementioned derivatives.

The macromolecule DNA (deoxyribonucleic acid) is the constituent ofchromosomes and different segments of this molecule form genes, whichare the supports of hereditary characteristics. DNA is in the form of adouble spiraled helix formed alternatively by sugar (deoxyribose) andphosphate, the spirals of the two chains being locally joined by groupsof purine or pyrimidine-type, nitrogen nucleic bases. The nucleotidesconstituting DNA are phosphoric esters of nucleosides.

The nucleic bases of DNA of an individual (or an animal orvegetable/plant) can be modified and damaged when said individual isexposed to intense solar radiation, cosmic radiation (intercontinentalflights), photosensitizers, contact with asbestos or ionizing radiation,no matter whether the latter is accidental or due to a radiotherapytreatment. These modifications of the nucleic bases of DNA can lead to asignificant change to the genetic patrimony of the individual inquestion. It is particularly important to detect if such modificationsare produced and to define the nature of the modifications which haveoccurred.

It would therefore be of interest to develop a determination or assayingprocedure usable in patients who may have undergone DNA modifications.

Among the various assaying procedures making it possible to determinethe presence of modified DNA in a sample, immunoassays consist ofreacting a specific antibody of a particular DNA modification with asample containing isolated or hydrolyzed DNA. These antibodies aregenerally produced by cloning.

The production of polyclonal or monoclonal antibodies firstly requiresthe production of specific antigens, i.e. nucleosides or nucleotides,whose nucleic bases have undergone the modifications which it is wishedto detect, said nucleotides or nucleotides being connected to a largemolecule, e.g. a protein. A nucleoside or nucleotide alone would be toosmall to be seen by the immune system. The polyclonal antibodies arethen produced by a mammal which has received an injection of the saidantigen, which contains a protein foreign to the mammal in question,linked with a hapten (small molecule against which it is wished toobtain the specific antibodies).

The prior art discloses methods for the immunological determination ofnucleic acids. The article by Christopher P. WILD: "Antibodies to DNAalkylation adducts as analytical tools in chemical carcinogenesis", Mut.Res., 1990, 233, pp 219-233, is devoted to specific antibodies ofnucleotides, whose nucleic bases have undergone modifications byalkylation. This author insists on the function of the antibodies in theimmunological determinations used in epidemiological studies of humancancers and chemical carcinogenesis, due in particular to alkylatingagents.

The article by B D. Stollar: "Immunochemical analyses of nucleic acids",Progress in Nucleic Acid, Research and Molecular Biology, 1992, 42, pp39-75 also relates to the specific antibodies of nucleic acids.

Certain oxidative deficiencies of DNA have also formed the subjectmatter of various publications.

Thus, the article by G. J. West et al: "Radioimmunoassay of8-hydroxyadenine" Int. J. Rad. Biol., 1982, 42, pp 481-490 describesradioimmunoassays of 8-hydroxyadenine. This DNA modification can arisefollowing irradiation with rays.

The article by P. Degan et al "Quantitation of8-hydroxy-2'-deoxyguanosine in DNA by polyclonalantibodies",Carcinogenesis, 1991, 12, pp 865-871 describes specific polyclonalantibodies of 8-hydroxy-2'-deoxyguanosine and 8-hydroguanine. Thesepolyclonal antibodies can be used in immunoassays in order to rapidlyisolate the two aforementioned types of modified guanosine, e.g. in aurine sample.

The article by H. L. Lewis et al "Serologic assay of DNA base damage",Rad. Res. 1978, 75 pp 305-316 describes the preparation of polyclonalantibodies and the assay of 5-hydroxymethyldeoxyuridine, obtained afterionizing radiation of thymidine.

Finally, the article by S. A. Leadon and P. C. Hanawalt, "Monoclonalantibody to DNA containing thymine glycol", Mut. Res., 1983, 112, pp191-200 deals with monoclonal antibodies of5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) obtained after the DNAhad been exposed to ionizing or near ultraviolet rays. These monoclonalantibodies were obtained by fusing myeloma cells of mice with spleencells from the BALB-c strain, the mice having been immunized with apoly(d-thymine) oxidized by OsO₄ and then complexed with methylatedbovine serum albumin. The tests were carried out by ELISA methods.

The article by B. F. Erlanger and S. M. Beiser "Antibodies specific forribonucleosides and ribonucleotides and their reaction with DNA", Proc.N.A.S., USA, 1964, 52, pp 68-74 describes a coupling protocol for anucleic acid with a carrier protein permitting the formation of anantigen usable in a rabbit immunization protocol. This coupling protocolis shown below. ##STR2##

R represents a purine or pyrimidine base, R' stands for H or --PO--(OH)₂and R" stands for the residue of a protein.

This process consists of acting on the monosaccharide of aribonucleoside by the action of sodium periodate. The monosaccharidecycle is open between carbons 2' and 3' and a dialdehyde is formed. Thisdialdehyde is then coupled to a protein at a pH close to 9 to 9.5. NaBH₄is used to reduce the Schiff's base obtained as the intermediate.

However, the process disclosed in said article is limited to nucleicbases which have not undergone modifications. Thus, with certain fragilebases sensitive to oxidation and/or reduction, it would be impossible tomodify beforehand these bases of nucleosides and then make said modifiednucleosides undergo an oxidation stage by sodium periodate, followed bya reduction stage using sodium hydridoborate. After such a chemicaltreatment, most of the defects for which it was wished to study modifiedDNA would have been deteriorated.

Moreover, the sequence of reactions described in this process takesplace without isolating the intermediates. Consequently a certain numberof parasitic products are liable to appear during the process and to bepresent in the final conjugate protein. Each parasitic product canpotentially induce an immune reaction inherent thereto. In this case,the mixture of antibodies obtained may only have a limited selectivity.This is the case in the article by H. L. Lewis et al referred tohereinbefore, where the target molecule was 5-hydroxymethyluracil andwhere the antiserum obtained also recognized thymine, which is a naturalbase of DNA.

The article by T. Okabayashi et al "A radioimmunoassay for1-β-D-Arabinofuranosylcytosine", Cancer Res., 1977, 17, pp 619-624discloses a process consisting of reacting a derivative of succinic acidwith a deoxynucleoside whose base has been modified, followed by thecreation of an amide function with the free amine of a protein. Thisprocess is illustrated below. ##STR3##

R represents a purine or pyrimidine base and R' the residue of aprotein.

The main defect of this process is the lack of stability of the esterfunction used for conjugating the nucleoside with the protein.

An article by M. D. Friesen et al "Isolation of urinary3-methyladenine", Chem. Res. Toxicol., 1991, 4, pp 102-106 discloses oneof the most widely used methods for coupling an alkylated nucleic baseto a protein and which is illustrated below. ##STR4##

In the above R is a protein and EDC represents 1-(3-dimethylamino)propyl!-3-ethyl dicarbodiimide hydrochloride.

This method differs from the two previous methods in that the startingproduct is neither a ribo- nor a deoxyribonucleoside in modified form,but is instead an alkylated derivative of the base. It makes it possibleto form antibodies of excellent quality, because the hapten used ispurified just prior to its conjugation with the protein. However, thisprocedure is more difficult to perform than the two methods describedhereinbefore.

The object of the invention is to develop antigens and processes for theproduction of said antigens avoiding the aforementioned disadvantages.

To this end, the invention relates to derivatives of nucleosides,characterized in that they comply with the following chemical formula:##STR5##

in which N is equal to one, R¹ stands for H or a linear mono-, di- ortri-phosphoric acid, R² is a hydroxyl group, an alkyl group, an arylgroup, a protein having an amino site, an aminoalkyl polystyrene or asilica grafted with an alkyl amine chain and R³ stands for a substitutedbase chosen from among uracil, thymine, cytosine, guanine or adenine.

The expression "derivatives of nucleosides" is used, because they areformed from nucleosides, but in said derivatives the monosaccharide hasdisappeared and is replaced by morpholine.

When R² represents a protein, the antigen obtained can serve as a basefor antibody production.

When R² is an aminoalkyl polystyrene or a grafted silica, e.g. theproduct obtained is a solid support to which is linked the hapten. Suchsupports can be used in ELISA (registered trademark)-typedeterminations.

The invention also relates to a process for the production ofderivatives of nucleosides complying with the following chemicalformula: ##STR6##

in which n represents a random positive integer, n is equal to 1, R¹stands for H or a linear mono-, di- or tri-phosphoric acid, R⁶ is ahydroxyl group, an alkyloxy group or an aryloxy group and R³ a modifiednucleic base chosen from among uracil, thymine, cytosine, guanine oradenine, consisting of reacting a substituent with a nucleosidederivative complying with the following general formula: ##STR7##

in which N is equal to 1, n represents a random positive integer, R¹ andR⁶ have the same meanings as hereinbefore and R⁴ stands for a nucleicbase chosen from among uracil, thymine, cytosine, guanine or adenine.

Unlike in the prior art processes, the process according to theinvention does not involve a fragile ester bond between the modifiednucleoside and the protein. Therefore the conjugates obtained are muchmore stable and can be stored in solution without undergoingdeterioration for a longer period.

The starting product (III) has a morpholine in place of thedeoxyribosidic cycle. This product is stable and is also carefullypurified prior to use in the process. This obviates the presence of"parasites" or contaminants in the hapten produced.

The starting product (III) has already undergone the transformation ofthe monosaccharide cycle into a morpholine cycle before the nucleic baseis modified in the process of the invention. This makes it possible toenvisage the preparation of antigens in which the nucleic bases areoxidized or reduced, which is much more difficult to obtain if thesynthesis of the morpholine cycle occurs after the modification of thenucleic base.

The invention also relates to another process for the production ofderivatives of nucleosides complying with the following chemicalformula: ##STR8##

in which n is equal to 1, n represents a random positive integer, R¹stands for H or a linear mon-, di- or tri-phosphoric acid, R³ stands fora modified nucleic base chosen from among uracil, thymine, cytosine,guanine or adenine and R⁵ represents the residue of a protein, an alkylpolystyrene or a silica grafted with an alkyl chain, said processconsisting of reacting a compound of type NH₂ --R⁵, in which R⁵ has thesame meaning as hereinbefore with a nucleoside derivative complying withthe following chemical formula: ##STR9##

in which n, R¹ and R³ have the same meanings as hereinbefore.

When NH₂ --R⁵ is a protein, the product (V) obtained is an antigen. WhenNH₂ --R⁵ is an aminoalkyl polystyrene or a silica grafted with an alkylamine chain, the product (V) obtained is a solid support. Thus, in anELISA determination process, the walls of the test tubes or thedepressions of the plates could be covered with grafted silica, whichcould permit the covalent bonding of the hapten to the support.

In addition, Pharmacia markets an apparatus for dynamically studying theinteractions between biological molecules (known under the registeredtrademark BiaCore). The principle consists of fixing one molecule to asolid support and circulating a solution containing the other. The solidsupport obtained according to the invention can be used in such anapparatus.

Moreover, the invention also relates to polyclonal antibodiesanti-derived from nucleosides, obtained by the immunization of asuitable mammal with the antigen complying with the following chemicalformula: ##STR10##

in which N is equal to 1, n represents a random positive integer, R¹represents H or a linear mono-, di- or tri-phosphoric acid, R³ standsfor a modified nucleic base chosen from among uracil, thymine, cytosine,guanine or adenine and R⁵ stands for a protein not obtained from saidmammal.

Finally, the invention relates to monoclonal antibodies anti-derivedfrom nucleosides obtained by fusing the myeloma cells of a mammal withthe spleen cells of mice immunized with an antigen complying with thefollowing chemical formula: ##STR11##

in which n is equal to 1, n represents a random positive integer, R¹stands for H or a linear mono-, di- or tri-phosphoric acid, R³ standsfor a modified nucleic base chosen from among uracil, thymine, cytosine,guanine or adenine and R⁵ stands for a protein not obtained from themouse.

The invention will be better understood from the reading of thefollowing description of an embodiment given in an exemplified andnon-limitative manner.

A first process for the preparation of derivatives of nucleosidesaccording to the invention consists of using a nucleoside or nucleosidealready modified by the formation of a morpholine, so as to permit thesubsequent bonding e.g. to an amino group belonging to a protein.

The starting product (III) (hereinafter called morpholinonucleoside) hasthe following chemical formula: ##STR12##

in which N is equal to 1, n represents a random positive integer, R¹stands for H or linear mono-, di- or tri-phosphoric acid, R⁶ stands fora hydroxyl group, an alkyloxy group or an aryloxy group and R⁴ standsfor a nucleic base chosen from among uracil, thymine, cytosine, guanineor adenine.

This product (III) can e.g. be obtained by a process described in thearticle by R. Rayford et al, "Reductive alkylation with oxidizednucleotides" J. Biol. Chem., 1985, 260, pp 15708-15713 and illustratedbelow: ##STR13##

R⁴ has the same meaning as hereinbefore.

This process consists of reacting a nucleoside with sodium periodate, inorder to open the bond between the 2' carbon and 3' carbon. This gives adialdehyde, which is reacted with glycine in order to form a doubleSchiff's base. This double base is reduced by NaCNBH₃ in order to obtaina morpholinonucleoside. The nucleoside used in the above article wasadenosine. However, in similar manner, this treatment can be performedwith other purine or pyrimidine bases. In the same way the nucleosidecan be replaced by a nucleotide in which the phosphoric acid is in the 5position.

When R⁶ is an alkyl or aryl group, the product (III) is an ester. It canbe obtained by the process described hereinbefore by replacing theglycine by a glycinate, such as e.g. a t-butyl or ethyl glycinate.Moreover, these derivatives can be transformed into active esters forcondensing to an amine function.

The starting product (III) of the process according to the invention canalso be obtained by any random other process.

The substituents making it possible to perform the substitutions on thepurine or pyrimidine bases are generally agents leading to oxidativemodifications of these bases. They are physical treatment or chemicalagents. The chemical agents are e.g. chosen from among ozone, hydrogenperoxide, bromine combined with collidine, bromine and silver oxide,potassium permanganate, osmium tetraoxide, methanal or AlLiH₄. The sameresult could be obtained by the action of ionizing rays in the presenceor absence of oxygen; by photochemistry (UV followed byphotosensitizer), by catalytic hydrogenation; or by treatment withbromine and water followed by hydrogenolysis. This is followed by asummary table I of substituents and the substitutions obtained by themon different purine or pyrimidine bases.

                                      TABLE I                                     __________________________________________________________________________    Nucleic bases  Substituent Substituted bases (R.sup.3)                        __________________________________________________________________________     ##STR14##     bromine, then collidine or ionizing rays or photochemistry                    (UV + photosensitizer)                                                                  s                                                                                ##STR15##                                                        ozone or ionizing rays or photochemistry (UV                                  + photosensitizer)                                                                      s                                                                                ##STR16##                                                        ionizing rays                                                                           s NHCONHCONH.sub.2                                                  or                                                                            photochemistry (UV +                                                          photosensitizer)                                                ##STR17##     bromine, then collidine or ionizing rays or photochemistry                    (UV + photosensitizer)                                                                     ##STR18##                                                        H.sub.2 CO                                                                                 ##STR19##                                          ##STR20##     Al.LiH.sub.4                                                                               ##STR21##                                          ##STR22##     ionizing rays or photochemistry (UV + photosensitizer)                                     ##STR23##                                                                     ##STR24##                                                        ozone       NHCHO                                                             or                                                                            ionizing rays                                                                 or                                                                            photochemistry (UV +                                                          photosensitizer)                                                              KM.sub.n O.sub.4 or O.sub.s O.sub.4  or Br.sub.2, then                        Ag.sub.2 O or ionizing rays or photochemistry (UV +                           photosensitizer)                                                                           ##STR25##                                                        hydrogen (by catalytic hydrogenation in the presence of                       palladium or rhodium) or photochemistry (UV                                   + photosensitizer)                                                                         ##STR26##                                                        or          or                                                                ionizing rays in the absence of oxygen                                                     ##STR27##                                          ##STR28##     H.sub.2 O.sub.2                                                                            ##STR29##                                                        Br.sub.2 + H.sub.2 O, then hydrogenolysis or ionizing rays                    r photochemistry (UV + photosensitizer)                                                    ##STR30##                                                        ionizing rays without oxygen or photochemistry (UV +                          photosensitizer)                                                                           ##STR31##                                          ##STR32##     Br.sub.2, then hydrogenolysis or ionizing                                                  ##STR33##                                                        ionizing rays without oxygen                                                               ##STR34##                                                        photochemistry (V + photosensitizer) or oxygen                                             ##STR35##                                                        rayonnements ionisants ou photochimie (U.V.                                   + phososensibilisateur)                                                                    ##STR36##                                         __________________________________________________________________________

At the end of the process according to the invention is obtained haptenwith the following chemical formula: ##STR37##

in which n is equal to 1, n represents a random positive integer, R¹stands for H or a linear mono-, di- or tri-phosphoric acid, R⁶ standsfor a hydroxyl group, an alkyloxy group or an aryloxy group and R³ is anucleic base chosen from among cytosine, uracil, thymine, adenosine andguanine and preferably one of the modified or substituted bases of tableI.

It should be noted that product II has a certain number of possiblevariants of product (I).

Hereinafter will be given a specific preparation example for one ofthese haptens.

EXAMPLE 1 Synthesis of2-(5-hydroxycytosin)-1-yl)-4-carboxymethyl-6-(hydroxymethyl)-morpholine

100 mg of 2-(cytosin-1-yl)-4-carboxymethyl-6-hydroxymethyl morpholineare dissolved in 5 ml of water. This is followed by the dropwiseaddition of bromine until the yellow colouring is maintained. After 15minutes stirring at ambient temperature, the excess bromine is expelledby bubbling air into the solution. This is followed by the addition of200 μl of collidine and the solution is stirred for 2 hours at ordinarytemperature. The water is evaporated under reduced pressure and then thefree collidine is eliminated by co-evaporation of 5 ml of ethanol. Theresidue obtained is analyzed by high pressure liquid chromatography(Nuclesil 10-C18 column of Macherey Nagel (registered trademark),dimensions 6×300 mm, mobile phase 50 mM triethyl ammonium acetate andmethanol). Analysis reveals the presence of a majority product (60%)different from the starting product. NMR analysis of the collectedproduct shows that it is indeed the expected compound. This product ischaracterized by an absorption maximum in the ultraviolet at 290.4 nmfor a molar extinction coefficient of 5700 mole/l⁻¹ cm⁻¹.

The second process of the invention consists of treating the hapten (VI)obtained at the end of the first process so as to e.g. form an antigenor solid phase (film, gel) in which the hapten VI is covalently fixed tothe support. This process is described below: ##STR38##

n, R¹ and R³ have the same meanings as hereinbefore and R⁵ representsthe residue of a protein, an alkyl polystyrene or a silica grafted withan alkyl chain.

The starting hapten (VI) is purified by high pressure liquidchromatography before being coupled to the radical NH₂ --R⁵. The radicalR⁵ can be a protein, which is e.g. chosen from among methylated bovineserum albumin, turkey or chicken egg albumin or hemocyanins and inparticular KLH or keyhole limpet helocyanin, i.e. a protein of thelimpet. This gives an antigen which can be used in the production ofantibodies. During the production of polyclonal antibodies, the proteinwill be chosen so as to be of a different nature from that of the animalused for antibody production. The aforementioned proteins are suitablewhen it is wished to immunize a rabbit.

The radical NH₂ --R⁵ can also be an aminoalkyl polystyrene or a silicagrafted with an alkyl amino chain. This gives a film or a gel in whichthe hapten is covalently bonded to the support.

It should be noted that product (V) has a certain number of possiblevariants of product (I).

An example of performing the process is given below.

EXAMPLE 2 Synthesis of a hapten-protein conjugate (antigen)

300 mg (90 μmoles) of2-(5-hydroxycytosin-1-yl)-4-carboxymethyl-6-hydroxymethylmorpholine aredissolved in 2 ml of water. This compound dissolved in water is added in3 hours to 5 ml of a solution containing 35 mg (180 μmoles) of E.D.C.(N'-(3-dimethylaminopropyl)-N-ethyl carbodiimide (hydrochloride)) and 80mg of bovine serum albumin. After allowing the solution to standovernight at ambient temperature, the water is evaporated under reducedpressure and the residue obtained is dissolved in 2 ml of water andanalyzed by exclusion chromatography (Fractogel TSK-HW40 (registeredtrademark) column of Merck, dimensions 20×400 mm, mobile phase 0.15MNaCl).

The product eluted first is collected, dialyzed against water and thesolution obtained is then lyophilized. The hapten charge of the productis then measured. This measurement is obtained by comparing the UVabsorption spectrum of the original protein with that of the proteingrafted on the modified nucleoside. The absorption comparison at 270 and300 nm makes it possible to establish that 7.8 moles of2-(5-hydroxycytosin-1-yl)-4-carboxymethyl-6-hydroxymethyl morpholine aregrafted per mole of protein.

The invention finally relates to the specific antibodies of antigens (V)obtained by the aforementioned process, in which R⁵ represents theresidue of a protein.

The polyclonal antibodies are obtained in conventional manner byinjection into rabbits, as described hereinafter. However, the antibodyobtained is novel, because it is specific to the novel, previouslyprepared antigen.

Hereinafter will be given an example of the preparation of thesepolyclonal antibodies.

EXAMPLE 3 Preparation of polyclonal antibodies specific to the antigenof example 2

The immunization protocol was based on two female rabbits of strain NZ"S.S.C." weighing 2 kg and which were treated in the manner describedhereinafter.

An emulsion was prepared by mixing 1 mg/liter of the hapten-proteinconjugate previously prepared in example 2, with 1 ml of complete Freundadjuvant. Each female rabbit received 1 injections of 100 μl of theaforementioned emulsion in the neck and back. Four weeks after the firstinjections the rabbits received a first booster under identicalconditions. After a further four week interval, they received a furtherbooster. The latter took place by intramuscular injection into the hipof 0.5 ml of an emulsion formed by 1 mg/ml of hapten-protein conjugateand 1 ml of incomplete Freund adjuvant.

Two weeks later 2 to 5 ml of blood were taken from the rabbit's ear. Thecollected serum made it possible to perform the first tests. Finally,four weeks after the second booster, a third booster was given byintramuscular injection using the same principle as for the secondbooster. Two weeks later the rabbits were killed and the maximum oftheir blood was collected, the serum containing the specific antibodiesof the aforementioned antigen.

The monoclonal antibodies can be obtained in conventional manner byfusing myeloma cells of a mammal such as a mouse with spleen cells e.g.from the BALB/c mouse strain, said mice having been immunized withantigen (V) constituted by modified morpholino nucleoside bonded to thecarrier protein (R⁵ representing a protein). These antibodies arespecific to the novel antigens obtained by the process according to theinvention.

Hereinafter is given an example of the preparation of these monoclonalantibodies.

EXAMPLE 4 Preparation of monoclonal antibodies specific to the antigenof example 2 (2-(5-hydroxycytosin-1-yl)-4-carboxylmethyl-6-hydroxymethylmorpholine)

Immunization:

A suspension of the antigen of example 2 was emulsified in an identicalvolume of complete Freund adjuvant. This emulsion was injectedintraperitoneally into female mice of strain BALB/c, aged 6 to 8 weeksand at a dosage rate of 0.1 ml of emulsion. Three weeks later, anintraperitoneal booster injection was given with an emulsion of theantigen of example 2 and incomplete Freund adjuvant. Two weeks afterthis booster, the final immunization took place by injection into theveins of the tail of the mouse, said injection incorporating the antigenof example 2 dissolved in 0.15M NaCl.

Cellular fusion and cloning

Three days after the final booster injection, the spleens were removedfrom the mice and pulverized. The spleen cells were washed in modifiedDulbecco medium not containing serum (DMEM). 10⁸ spleen cells were mixedwith 10⁷ myeloma cells and then centrifuged at 500×g for 7 minutes atambient temperature. This was followed by the elimination of the mediumand the recovery of the centrifuging mass, to which was added 0.8 ml of50% PEG 4000 (Merck), over a period of 1 minute accompanied by gentlestirring at 37° C. This was followed by the addition of modifiedDulbecco medium at a rate of 1 ml in 1 minute, then 20 ml for 5 minutes.The cells were centrifuged at 200×g for 10 minutes and then thecentrifuging mass was resuspended in 15 ml of DMEM and 10% FBS (fetalbovine serum). 0.05 ml aliquot portions were distributed in platesproided with culture depressions coated with macrophages. The plateswere allowed to incubate for 24 hours at 37° C. before adding to thedepressions hypoxanthine (1.10⁻⁴ M), amethopterin (4.10⁻⁷ M) andthymidine (1.6₄ 10⁻⁵ M) in DMEM (HAT medium). Seven days after fusing,addition took place of 0.025 ml of HAT medium to each depression and themedium was then replenished every 3 to 4 days. The colonies were testedby the ELISA method for their activity against the antigen of example 2.Only the cells corresponding to the positive depressions were cloned.

We claim:
 1. A nucleoside derivative having the structure of chemicalformula (I): ##STR39## wherein n is equal to one; R¹ is a hydrogen or alinear mono-, di-, or tri-phosphoric acid; R² is selected from the groupconsisting of hydroxyl, alkyloxy, aryloxy, a protein comprising a fleeamino group, an amino-alkyl polystyrene, and a silica grafted to analkyl amine chain; and R³ is selected from the group, consisting of(5-hydroxycytosin)-1-yl, (5-hydroxyhydantoin)-1-yl,--NH--CO--NH--CO--NH₂, (5-hydroxyuracil)-1-yl, (5-formyluracil)-1-yl,(5-hydroxymethyluracil)-1-yl, --NH--CHO, (5,6-dihydroxythymin)-1yl,(5,6-dihydrothymin)-1-yl, (5,6-dihydro-5-hydroxythymin)-1-yl,adenine-Nl-oxide, (8-oxo-7,8-dihydroadenin)-9-yl,(6-amino-5-formylamine-pyrimindin)-4-yl!amino,(8-oxo-7,8-dihydroguanin)-9-yl,(2-amino-6-oxo-5-formylamino-pyrimidin)-4-yl!amino,(4,8-dihydro-4-hydroxy-8-oxo-guanin)-9-yl, and(2,2-diaminooxazol-4-one)-5-yl!amino.
 2. The nucleoside derivativeaccording to claim 1, wherein the protein is selected from the groupconsisting of methylated bovine serum albumin, turkey albumin, chickenegg albumin, and hemocyanins.
 3. A process for the preparation of anucleoside derivative having the structure of chemical formula (II):##STR40## wherein n is equal to one; R¹ is a hydrogen or a linear mono-,di- or tri-phosphoric acid; R⁶ is a hydroxyl group, an alkyl group or anaryl group; and R³ is a substituted heterocyclic base wherein theheterocyclic base is selected from the group consisting of uracil,thymine, cytosine, guanine, and adenine, said process comprisingtreating a nucleoside derivative having the structure of chemicalformula (III): ##STR41## wherein n is a equal to 1; R¹ is a hydrogen ora linear mono-, di-, or tri-phosphoric acid; R⁶ is a hydroxyl group, analkyl group, or an aryl group; and R⁴ is a heterocyclic base selectedfrom the group consisting of uracil, thymine, cytosine, guanine, andadenine, with a chemical reaction-inducing reagent.
 4. The process asclaimed in claim 3, wherein the chemical reaction-inducing reagent isselected from the group consisting of ozone, hydrogen peroxide, brominecombined with collidine, bromine and Ag₂ O, potassium permanganate,osmium, tetraoxide, methanal and AlLiH₄.
 5. The process as claimed inclaim 3, wherein the chemical reaction-inducing reagent is selected fromthe group consisting of ultraviolet radiation followed by aphotosensitizer; ionizing rays in the presence or absence of oxygen;catalytic hydrogenation; and treatment with bromine and water followedby hydrogenolysis.
 6. The process as claimed in claim 3, wherein R³ isselected from the group consisting of (5-hydroxycytosin)-1-yl,(5-hydroxyhydantoin)-1-yl, --NH--CO--NH--CO--NH₂,(5-hydroxyuracil)-1-yl, (5-formyluracil)-1-yl,(5-hydroxymethyluracil)-1-yl, --NH--CHO,(5,6-dihydro-5,6-dihydro-5,6-dihydroxythymin)-1-yl,(5,6-dihydrothymin)-1-yl, (5,6-dihydro-5-hydroxythymin)-1-yl,adenine-Nl-oxide, (8-oxo-7,8-dihydroadenine)-9-yl,(6-amino-5-formylamine-pyrimidin)-4-yl!-amino,(8-oxo-7,8-dihydroguanin)-9-yl,(2-amino-6-oxo-5-formylamino-pyrimidin)-4-1!-amino,(4,8-dihydro-4-hydroxy-8-oxo-guanin)-9-yl and(2,2-diamino-oxazol-4-one)-5-yl!-amino!.
 7. A process for thepreparation of a nucleoside derivative having the structure of chemicalformula (V): ##STR42## wherein n is equal to 1; R¹ is a hydrogen or alinear mono-, di-, or tri-phosphoric acid; R⁵ is selected from a groupconsisting of a residue of a protein, an alkyl polystyrene, or a silicagrafted to an alkyl chain; and R³ is a substituted heterocyclic basewherein the heterocyclic base is selected from the group consisting ofuracil, thymine, cytosine, guanine, and adenine, said process comprisingreacting a compound of chemical structure NH₂ --R⁵, wherein R⁵ isselected from the group consisting of a residue of a protein, an alkylpolystyrene, or a silica grafted to an alkyl chain, with a nucleosidederivative having the structure of chemical formula (VI): ##STR43##wherein n is equal to 1; R¹ is a hydrogen or a linear mono-, di-, ortri-phosphoric acid; and R³ is a heterocyclic base selected from thegroup consisting of uracil, thymine, cytosine, guanine, and adenine. 8.The process as claimed in claim 7, wherein R³ is selected from the groupconsisting of (5-hydroxycytosin)-1-yl, (5-hydroxyhydantoin)-1-yl,--NH--CO--NH--CO--NH₂, (5-hydroxyuracil)-1-yl, (5-formyluracil)-1-yl,(5-hydroxy-methyluracil)-1-yl, --NH--CHO,(5,6-dihydro-5,6-dihydro-5,6-dihydroxythymin)-1-yl,(5,6-dihydrothymin)-1-yl, (5,6-dihydro-5-hydroxythymin)-1-yl,adenine-Nl-oxide, (8-oxo-7,8-dihydroadenine)-9-yl,(6-amino-5-formylamine-pyrimidin)-4-yl!-amino,(8-oxo-7,8-dihydroguanin)-9-yl,(2-amino-6-oxo-5-formylamino-pyrimidin)-4-1!-amino,(4,8-dihydro-4-hydroxy-8-oxo-guanin)-9-yl and(2,2-diamino-oxazol-4-one)-5-yl!-amino!.
 9. A polyclonal antibodyproduced by immunizing a mammal with an antigen having the structure ofchemical formula (V): ##STR44## wherein n is equal to 1; R¹ is ahydrogen or a linear mono-, di-, or tri-phosphoric acid; R³ is selectedfrom the group consisting of (5-hydroxycytosin)-1-yl,(5-hydroxyhydantoin)-1-yl, --NH--CO--NH--CO--NH₂,(5-hydroxyuracil)-1-yl, (5-formyluracil)-1-yl,(5-hydroxymethyluracil)-1-yl, --NH--CHO,(5,6-dihydro-5,6-dihydro-5,6-dihydroxythymin)-1-yl,(5,6-dihydrothymin)-1-yl, (5,6-dihydro-5-hydroxythymin)-1-yl,adenine-Nl-oxide, (8-oxo-7,8-dihydroadenine)-9-yl,(6-amino-5-formylamine-pyrimidin)-4-yl!-amino,(8-oxo-7,8-dihydroguanin)-9-yl,(2-amino-6-oxo-5-formylamino-pyrimidin)-4-1!-amino,(4,8-dihydro-4-hydroxy-8-oxo-guanin)-9-yl and(2,2-diamino-oxazol-4-one)-5-yl!-amino; and R⁵ consists of a protein.10. The polyclonal antibody according to claim 9, wherein R⁵ is aprotein selected from the group consisting of methylated bovine serumalbumin, turkey albumin, chicken egg albumin, and hemocyanins.
 11. Amonoclonal antibody produced by the process of fusing myeloma cells of amammal with spleen cells of a mouse immunized with an antigen having thestructure of chemical formula (V): ##STR45## wherein n is equal to 1; R¹is a hydrogen or a linear mono-, di-, or tri-phosphoric acid; R³ isselected from the group consisting of (5-hydroxycytosin)-1-yl,(5-hydroxyhydantoin)-1-yl, --NH--CO--NH--CO--NH₂,(5-hydroxyuracil)-1-yl, (5-formyluracil)-1-yl,(5-hydroxymethyluracil)-1-yl, --NH--CHO,(5,6-dihydro-5,6-dihydro-5,6-dihydroxythymin)-1-yl,(5,6-dihydrothymin)-1-yl, (5,6-dihydro-5-hydroxythymin)-1-yl,adenine-Nl-oxide, (8-oxo-7,8-dihydroadenine)-9-yl,(6-amino-5-formylamine-pyrimidin)-4-yl!-amino,(8-oxo-7,8-dihydroguanin)-9-yl(2-amino-6-oxo-5-formylamino-pyrimidin)-4-1!-amino,(4,8-dihydro-4-hydroxy-8-oxo-guanin)-9-yl and2,2-diamino-oxazol-4-one)-5-yl!-amino; and R⁵ consists of a protein. 12.The monoclonal antibody according to claim 11, wherein R⁵ is a proteinselected from the group consisting of methylated bovine serum albumin,chicken albumin, turkey egg albumin, and hemocyanins.